Bactericidal/permeability-increasing protein (BPI) deletion analogs

ABSTRACT

Novel BPI deletion analogs are provided that consist of amino acid residues 10 through 193 of mature human BPI wherein the cysteine residue at BPI amino acid position 132 is replaced by another amino acid. Fusion proteins comprising these analogs are also provided, as are polynucleotides encoding these products, materials and methods for their recombinant production, compositions and medicaments of these products, and therapeutic uses for these products.

This application is a continuation-in-part of U.S. Ser. No. 09/099,725filed Jun. 19, 1998 now U.S. Pat. No. 6,013,631.

BACKGROUND OF THE INVENTION

The present invention provides preparations of novel biologically activedeletion analogs of bactericidal/permeability-increasing protein (BPI)characterized by improved stability and homogeneity as well as byenhanced in vivo activity, and pharmaceutical compositions containingthe same.

BPI is a protein isolated from the granules of mammalianpolymorphonuclear leukocytes (PMNs or neutrophils), which are bloodcells essential in the defense against invading microorganisms. BPI isknown to bind to lipopolysaccharide, a major component of the outermembrane of gram-negative bacteria that stimulates a potent inflammatoryresponse which can lead to septic shock. Human BPI protein has beenisolated from PMNs by acid extraction combined with either ion exchangechromatography [Elsbach, J. Biol. Chem., 254:11000 (1979)] or E. coliaffinity chromatography [Weiss, et al., Blood, 69:652 (1987)]. BPIobtained in such a manner is referred to herein as natural BPI and hasbeen shown to have potent bactericidal activity against a broad spectrumof gram-negative bacteria. The molecular weight of human BPI isapproximately 55,000 daltons (55 kD). The amino acid sequence of theentire human BPI protein and the nucleic acid sequence of DNA encodingthe protein have been reported in FIG. 1 of Gray et al., J. Biol. Chem.,264:9505 (1989), incorporated herein by reference. The Gray et al. aminoacid sequence is set out in SEQ ID NO: 1 hereto. U.S. Pat. No.5,198,541, the disclosure of which is incorporated herein by reference,discloses recombinant genes encoding, and methods for expression of, BPIproteins including recombinant BPI holoprotein, referred to as rBPI, andrecombinant fragments of BPI.

A proteolytic N-terminal fragment of BPI having a molecular weight ofabout 25 kD has an amphipathic character, containing alternatinghydrophobic and hydrophilic regions. This N-terminal fragment of humanBPI possesses the anti-bacterial activity of the naturally-derived 55 kDhuman BPI holoprotein. [Ooi et al., J. Bio. Chem., 262: 14891-14894(1987)]. In contrast to the N-terminal portion, the C-terminal region ofthe isolated human BPI protein displays only slightly detectableanti-bacterial activity against gram-negative organisms. [Ooi et al., J.Exp. Med., 174:649 (1991).] An N-terminal BPI fragment of approximately23 kD, referred to as "rBPI₂₃," has been produced by recombinant meansand also retains anti-bacterial activity against gram-negativeorganisms. [Gazzano-Santoro et al., Infect. Immun. 60:4754-4761 (1992).]An N-teminal analog of BPI, rBPI₂₁, has been produced as described inHorwitz et al., Protein Expression Purification, 8:28-40 (1996).

The bactericidal effect of BPI has been reported to be highly specificto gram-negative species, e.g., in Elsbach and Weiss, Inflammation:Basic Principles and Clinical Correlates, eds. Gallin et al., Chapter30, Raven Press, Ltd. (1992). This reported target cell specificity wasbelieved to be the result of the strong attraction of BPI forlipopolysaccharide (LPS), which is unique to the outer membrane (orenvelope) of gram-negative organisms. Although BPI was commonly thoughtto be non-toxic for other microorganisms, including yeast, and forhigher eukaryotic cells, it has recently been discovered, as discussedinfra, that BPI protein products, exhibit activity against gram-positivebacteria, mycoplasma, mycobacteria, fungi, protozoa, and chlamydia.

The precise mechanism by which BPI kills gram-negative bacteria is notyet completely elucidated, but it is believed that BPI must first bindto the surface of the bacteria through electrostatic and hydrophobicinteractions between the cationic BPI protein and negatively chargedsites on LPS. LPS has been referred to as "endotoxin" because of thepotent inflammatory response that it stimulates, i.e., the release ofmediators by host inflammatory cells which may ultimately result inirreversible endotoxic shock. BPI binds to lipid A, reported to be themost toxic and most biologically active component of LPS.

In susceptible gram-negative bacteria, BPI binding is thought to disruptLPS structure, leading to activation of bacterial enzymes that degradephospholipids and peptidoglycans, altering the permeability of thecell's outer membrane, and initiating events that ultimately lead tocell death. [Elsbach and Weiss (1992), supra]. BPI is thought to act intwo stages. The first is a sublethal stage that is characterized byimmediate growth arrest, permeabilization of the outer membrane andselective activation of bacterial enzymes that hydrolyze phospholipidsand peptidoglycans. Bacteria at this stage can be rescued by growth inserum albumin supplemented media [Mannion et al., J. Clin. Invest.,85:853-860 (1990)]. The second stage, defined by growth inhibition thatcannot be reversed by serum albumin, occurs after prolonged exposure ofthe bacteria to BPI and is characterized by extensive physiologic andstructural changes, including apparent damage to the inner cytoplasmicmembrane.

Initial binding of BPI to LPS leads to organizational changes thatprobably result from binding to the anionic groups of LPS, whichnormally stabilize the outer membrane through binding of Mg⁺⁺ and Ca⁺⁺.Attachment of BPI to the outer membrane of gram-negative bacteriaproduces rapid permeabilization of the outer membrane to hydrophobicagents such as actinomycin D. Binding of BPI and subsequentgram-negative bacterial killing depends, at least in part, upon the LPSpolysaccharide chain length, with long O-chain bearing, "smooth"organisms being more resistant to BPI bactericidal effects than shortO-chain bearing, "rough" organisms [Weiss et al., J. Clin. Invest. 65:619-628 (1980)]. This first stage of BPI action, permeabilization of thegram-negative outer envelope, is reversible upon dissociation of theBPI, a process requiring high concentrations of divalent cations andsynthesis of new LPS [Weiss et al., J. Immunol. 132: 3109-3115 (1984)].Loss of gram-negative bacterial viability, however, is not reversed byprocesses which restore the envelope integrity, suggesting that thebactericidal action is mediated by additional lesions induced in thetarget organism and which may be situated at the cytoplasmic membrane(Mannion et al., J. Clin. Invest. 86: 631-641 (1990)). Specificinvestigation of this possibility has shown that on a molar basis BPI isat least as inhibitory of cytoplasmic membrane vesicle function aspolymyxin B (In't Veld et al., Infection and Immunity 56: 1203-1208(1988)) but the exact mechanism as well as the relevance of suchvesicles to studies of intact organisms has not yet been elucidated.

BPI protein products (which include naturally and recombinantly producedBPI protein; natural, synthetic, and recombinant biologically activepolypeptide fragments of BPI protein; biologically active polypeptidevariants of BPI protein or fragments thereof, including hybrid fusionproteins and dimers; biologically active polypeptide analogs of BPIprotein or fragments or variants thereof, including cysteine-substitutedanalogs; and BPI-derived peptides) have been demonstrated to have avariety of beneficial activities. BPI protein products are known to bebactericidal for gram-negative bacteria, as described in U.S. Pat. Nos.5,198,541 and 5,523,288, both of which are incorporated herein byreference. BPI protein products are also known to enhance theeffectiveness of antibiotic therapy in gram-negative bacterialinfections, as described in U.S. Pat. No. 5,523,288 and correspondingInternational Publication No. WO 95/08344 (PCT/US94/11225), which areincorporated herein by reference. BPI protein products are also known tobe bactericidal for gram-positive bacteria and mycoplasma, and toenhance the effectiveness of antibiotics in gram-positive bacterialinfections, as described in U.S. Pat. No. 5,578,572 and correspondingInternational Publication No. WO 95/19180 (PCT/US95/00656), which areincorporated herein by reference. BPI protein products are further knownto exhibit anti-fungal activity, and to enhance the activity of otheranti-fungal agents, as described in U.S. Pat. No. 5,627,153 andcorresponding International Publication No. WO 95/19179(PCT/US95/00498), and further as described for anti-fungal peptides inco-owned, co-pending U.S. application Ser. No. 08/621,259 filed Mar. 21,1996, which is in turn a continuation-in-part of U.S. application Ser.No. 08/504,841 filed Jul. 20, 1994 and corresponding InternationalPublication Nos. WO 96/08509 (PCT/US95/09262) and WO 97/04008(PCT/US96/03845), all of which are incorporated herein by reference. BPIprotein products are further known to exhibit anti-protozoan activity,as described in U.S. Pat. No. 5,646,114 and corresponding InternationalPublication No. WO 96/01647 (PCT/US95/08624), all of which areincorporated herein by reference. BPI protein products are known toexhibit anti-chlamydial activity, as described in co-owned, co-pendingU.S. application Ser. No. 08/694,843 filed Aug. 9, 1996 andcorresponding International Publication No. WO 98/06415(PCT/US97/13810), all of which are incorporated herein by reference.Finally, BPI protein products are known to exhibit anti-mycobacterialactivity, as described in co-owned, co-pending U.S. application Ser. No.08/626,646 filed Apr. 1, 1996, which is in turn a continuation of U.S.application Ser. No. 08/285,803 filed Aug. 14, 1994, which is in turn acontinuation-in-part of U.S. application Ser. No. 08/031,145 filed Mar.12, 1993 and corresponding International Publication No. W094/20129(PCT/US94/02463), all of which are incorporated herein by reference.

The effects of BPI protein products in humans with endotoxin incirculation, including effects on TNF, IL-6 and endotoxin are describedin U.S. Pat. Nos. 5,643,875 and 5,753,620 and correspondingInternational Publication No. WO 95/19784 (PCT/US95/01151), all of whichare incorporated herein by reference.

BPI protein products are also known to be useful for treatment ofspecific disease conditions, such as meningococcemia in humans (asdescribed in co-owned, co-pending U.S. application Ser. No. 08/644,287filed May 10, 1996 and corresponding International Publication No. WO97/42966 (PCT/US97/08016), which are incorporated herein by reference),hemorrhagic trauma in humans, (as described in co-owned, co-pending U.S.application Ser. No. 08/862,785, a continuation-in-part of U.S. Ser. No.08/652,292 filed May 23, 1996, now U.S. Pat. No. 5,756,464, andcorresponding International Publication No. WO 97/44056(PCT/US97/08941), all of which are incorporated herein by reference),burn injury (as described in U.S. Pat. No. 5,494,896 and correspondingInternational Publication No. WO 96/30037 (PCT/US96/02349), both ofwhich are incorporated herein by reference), ischemia/reperfusion injury(as described in U.S. Pat. No. 5,578,568, incorporated herein byreference), and liver resection (as described in co-owned, co-pendingU.S. application Ser. No. 08/582,230 filed Mar. 16, 1998 which is acontinued prosecution application of the same serial no. filed Jan. 3,1996, which is in turn a continuation of U.S. application Ser. No.08/318,357 filed Oct. 5, 1994, which is in turn a continuation-in-partof U.S. application Ser. No. 08/132,510 filed Oct. 5, 1993, andcorresponding International Publication No. WO 95/10297(PCT/US94/11404), all of which are incorporated herein by reference).

BPI protein products are also known to neutralize the anti-coagulantactivity of exogenous heparin, as described in U.S. Pat. No. 5,348,942,incorporated herein by reference, as well as to be useful for treatingchronic inflammatory diseases such as rheumatoid and reactive arthritis,as described in U.S. Pat. No. 5,639,727, incorporated herein byreference, and for inhibiting angiogenesis and for treatingangiogenesis-associated disorders including malignant tumors, ocularretinopathy and endometriosis, as described in co-owned, co-pending U.S.application Ser. Nos. 08/435,855, 08/466,624 and 08/466,826, andcorresponding International Publication No. WO 94/20128(PCT/US94/02401), all of which are incorporated herein by reference.

BPI protein products are also known for use in antithrombotic methods,as described in U.S. Pat. No. 5,741,779 and corresponding InternationalPublication No. W097/42967 (PCT/US97/08017), which are incorporatedherein by reference.

U.S. Pat. Nos. 5,420,019 and 5,674,834 and corresponding InternationalPublication No. W094/18323 (PCT/US94/01235), all of which areincorporated herein by reference, discloses that the replacement of thecysteine residue at amino acid position 132 or 135 with another aminoacid renders the resulting BPI polypeptide resistant to dimerization andcysteine adduct formation. It also discloses that terminating theN-terminal BPI fragment at BPI amino acid position 193 resulted in anexpression product with reduced carboxy-terminal heterogeneity.

Of interest is the report in Capodici and Weiss, J. Immunol.,156:4789-4796 (1996) that the in vitro transcription/translationproducts of DNA encoding amino acid residues 1 through 193 (BPI₁₋₁₉₃)and residues 13 through 193 (BPI₁₃₋₁₉₃) of mature BPI showed similarLPS-dependent binding to immobilized LPS.

There continues to be a need in the art for improved biologically activeBPI protein product preparations, particularly those with enhancedstability, homogeneity and/or in vivo biological activity.

SUMMARY OF THE INVENTION

The present invention provides novel biologically active BPI deletionanalogs and preparations thereof characterized by enhanced stability andhomogeneity, including for example, resistance to dimerization andcysteine adduct formation and reduced amino-terminal andcarboxy-terminal heterogeneity of the recombinant product, as well as byenhanced in vivo biological activity, properties which render it highlysuitable for therapeutic and diagnostic uses. Novel BPI deletion analogsare the expression product of DNA encoding amino acid residues 10through 193 of mature human BPI (SEQ ID NO: 2), in which the cysteine atposition 132 has been replaced with a different amino acid, preferably anon-polar amino acid such as serine or alanine. In a preferredembodiment, designated "rBPI(10-193)C132A" or "rBPI(10-193)ala¹³²," thecysteine at position 132 is replaced with an alanine.

The invention further provides novel purified and isolatedpolynucleotide sequences (e.g., DNA or RNA) encoding these BPI proteinproducts; materials and methods for their recombinant production,including vectors and host cells comprising the DNA; improved stablepharmaceutical compositions comprising these BPI protein products; andimproved treatment methods using these compositions, either alone orconcurrently administered with other therapeutic agents. Alsocontemplated is the use of the BPI deletion analogs of the invention inmanufacture of a medicament for treating a subject that would benefitfrom administration of BPI protein product.

Numerous additional aspects and advantages of the invention will becomeapparent to those skilled in the art upon considering the followingdetailed description of the invention, which describes the presentlypreferred embodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the elevation in blood pressure, measured as area underthe curve (AUC) occurring after administration of eitherrBPI(10-193)C132A or rBPI₂₁.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel BPI deletion analogs consisting ofamino acid residues 10 through 193 of mature human BPI (set forth in SEQID NO: 2) wherein the cysteine residue at BPI amino acid position 132 isreplaced by another amino acid, preferably a non-polar amino acid suchas serine or alanine. A preferred embodiment, in which the cysteine atposition 132 is replaced with an alanine, has been designatedrBPI(10-193)C132A or rBPI(10-193)ala¹³².

The BPI protein product rBPI₂₁ is the expression product of DNA encodingamino acid residues 1 to 193 of mature human BPI wherein the cysteine atresidue number 132 is substituted with alanine, described in U.S. Pat.No. 5,420,019. Changes in the fermentation processes used to producerBPI₂₁ by recombinant methods that achieved higher cell densities andhigher rBPI₂₁ titers also resulted in an apparent increase inamino-terminal heterogeneity of the purified product. In somefermentation runs, up to about 20 % of the purified product was observedto be a species with amino acids 10-193 of BPI, rather than the encoded1-193 amino acids. SDS-PAGE gels of 500-liter fermentor samples over thecourse of a fermentation run showed that this 10-193 species appeared inthe last 2-3 days of the run, with the greatest amount appearing on theday of harvest. Further investigation revealed that incubation of rBPI₂₁with a CHO-K1 cell homogenate yielded a digested product, suggestingthat protease activity associated with the cells was involved. Tosimulate protease activity in a controlled manner, rBPI₂₁ was incubatedwith aminopeptidase M and elastase. The rBPI₂₁ was resistant toaminopeptidase M digestion, but elastase rapidly converted the rBPI₂₁into 40 % BPI(8-193) and 60% BPI(10-193).

As described herein, stable homogeneous preparations ofrBPI(10-193)C132A were produced proteolytically and by recombinantmethods. The protein was purified and was tested for biologicalactivity. Experiments were performed to compare rBPI(10-193)C132A torBPI₂₁ in several in vitro biological assays, two different animalefficacy models and in pharmacokinetic and toxicology studies. Asdescribed in Examples 5-7, rBPI(10-193)C132A and rBPI₂₁ had similar invitro activities when compared in radial diffusion and brothmicrodilution bactericidal assays with Escherichia coli J5, a radialdiffusion assay with an L-form of Staphylococcus aureus, a competitionbinding assay with E. coli J5 LPS, and in LPS neutralization assays withRAW and THP1 cells. Additional experiments described in Example 5 showedthat rBPI(10-193)C132A appeared to be approximately twice as potent asrBPI₂₁ in an LPS binding assay using rate nephelometry. As described inExample 8, purified rBPI(10-193)C132A and rBPI₂₁ had similar toxicityprofiles in a GLP toxicology study in rats at doses up to 120 mg/kg/dayfor three days and similar pharmacokinetics in rats at a dose of 2mg/kg. Experiments described in Example 8 also showed that in a mouseendotoxin challenge model, rBPI(10-193)C132A appeared to be at leasttwo-fold more potent than rBPI₂₁ in two studies whereas in a mouse modelof lethal bacteremia, rBPI(10-193)C132A and rBPI₂₁ were similarlypotent. In additional in vivo experiments in conscious rats, doses of 40and 50 mg/kg of infused rBPI₂₁ caused significant transient decreases inblood pressure relative to the vehicle control, while the same doses ofrBPI(10-193)C132A did not result in a statistically significanttransient decrease in blood pressure relative to control. Thus, infusionof rBPI(10-193)C132A appears to provide a reduction in an adverse effectin blood pressure compared with infusion of rBPI₂₁.

The invention further contemplates fusion of rBPI(10-193)C132A with atleast a portion of at least one other polypeptide. Examples of suchhybrid fusion proteins are described in U.S. Pat. No. 5,643,570 andcorresponding International Publication No. WO 93/23434(PCT/US93/04754), which are all incorporated herein by reference andinclude hybrid fusion proteins comprising, at the amino-terminal end, aBPI protein or a biologically active fragment thereof and, at thecarboxy-terminal end, at least one constant domain of an immunoglobulinheavy chain or allelic variant thereof.

The invention additionally contemplates purified and isolatedpolynucleotide sequences (e.g., DNA or RNA) encoding the novel BPIdeletion analogs or fusion proteins of the present invention; expressionvectors containing such polynucleotides, preferably operatively linkedto an endogenous or heterologous expression control sequence;prokaryotic or eukaryotic host cells stably transfected or transformedwith a DNA or vector of the present invention; and methods for therecombinant production of the novel deletion analog BPI protein productsof the present invention, e.g., methods in which a host cell is grown ina suitable nutrient medium and the deletion analog BPI protein productis isolated from the cell or the medium. Such polynucleotide sequencesor vectors may optionally encode the 27-amino acid BPI leader sequenceand the mouse light chain polyadenylation signal.

The recombinantly produced novel BPI deletion analog of the presentinvention may be produced according to the methods described in U.S.Pat. No. 5,439,807 and corresponding International Publication No. WO93/23540 (PCT/US93/04752), which are all incorporated herein byreference. U.S. Pat. No. 5,439,807 discloses methods for thepurification of recombinant BPI protein products expressed in andsecreted from genetically transfected mammalian host cells in culture,and discloses how one may produce large quantities of recombinant BPIproducts suitable for incorporation into stable, homogeneouspharmaceutical preparations.

The present invention further provides improved stable pharmaceuticalcompositions comprising the novel BPI deletion analogs and improvedtreatment methods using these compositions, either alone or concurrentlyadministered with other therapeutic agents. It is contemplated that suchcompositions may be utilized in any of the therapeutic uses known forBPI protein products, including those discussed supra.

The administration of BPI protein products in general, including BPIdeletion analogs, is preferably accomplished with a pharmaceuticalcomposition comprising a BPI protein product and a pharmaceuticallyacceptable diluent, adjuvant, or carrier. The BPI protein product may beadministered without or in conjunction with known surfactants, otherchemotherapeutic agents or additional known anti-chlamydial agents. Astable pharmaceutical composition containing BPI protein products (e.g.,rBPI₂₃) comprises the BPI protein product at a concentration of 1 mg/mlin citrate buffered saline (5 or 20 mM citrate, 150 mM NaCl, pH 5.0)comprising 0.1% by weight of poloxamer 188 (Pluronic F-68, BASF,Parsippany, N.J.) and 0.002% by weight of polysorbate 80 (Tween 80, ICIAmericas Inc., Wilmington, Del. or JT Baker, Phillipsburg, N.J.).Another stable pharmaceutical composition containing BPI proteinproducts (e.g., rBPI₂₁) comprises the BPI protein product at aconcentration of 2 mg/ml in 5 mM citrate, 150 mM NaCl, 0.2% poloxamer188 and 0.002% polysorbate 80. Such preferred combinations are describedin U.S. Pat. Nos. 5,488,034 and 5,696,090 and correspondingInternational Publication No. WO 94/17819 (PCT/US94/01239), thedisclosures of all of which are incorporated herein by reference. Asdescribed in U.S. application Ser. No. 08/586,133 filed Jan. 12, 1996,which is in turn a continuation-in-part of U.S. application Ser. No.08/530,599 filed Sep. 19, 1995, which is in turn a continuation-in-partof U.S. application Ser. No. 08/372,104 filed Jan. 13, 1995, andcorresponding International Publication No. W096/21436 (PCT/US96/01095),all of which are incorporated herein by reference, other poloxamerformulations of BPI protein products with enhanced activity may beutilized.

Therapeutic compositions comprising BPI protein product may beadministered systemically or topically. Systemic routes ofadministration include oral and parenteral routes, includingintravenous, intramuscular or subcutaneous injection (including into adepot for long-term release), intraocular and retrobulbar, intrathecal,intraperitoneal (e.g. by intraperitoneal ravage), intrapulmonary (usingpowdered drug, or an aerosolized or nebulized drug solution), ortransdermal. Improved aerosolized formulations are described inco-owned, co-pending U.S. application Ser. No. 08/962,217 filed Oct. 31,1997 and corresponding International Publication No. WO 98/19694(PCT/US97/19850), which are both incorporated herein by reference.

When given parenterally, BPI protein product compositions are generallyinjected in doses ranging from 1 μg/kg to 100 mg/kg per day, preferablyat doses ranging from 0.1 mg/kg to 20 mg/kg per day, more preferably atdoses ranging from 1 to 20 mg/kg/day and most preferably at dosesranging from 2 to 10 mg/kg/day. The treatment may continue by continuousinfusion or intermittent injection or infusion, at the same, reduced orincreased dose per day for, e.g., 1 to 3 days, and additionally asdetermined by the treating physician. When administered intravenously,BPI protein products are preferably administered by an initial briefinfusion followed by a continuous infusion. The preferred intravenousregimen is a 1 to 20 mg/kg brief intravenous infusion of BPI proteinproduct followed by a continuous intravenous infusion at a dose of 1 to20 mg/kg/day, continuing for up to one week. A particularly preferredintravenous dosing regimen is a 1 to 4 mg/kg initial brief intravenousinfusion followed by a continuous intravenous infusion at a dose of 1 to4 mg/kg/day, continuing for up to 72 hours.

Topical routes include administration in the form of salves, creams,jellies, ophthalmic drops or ointments (as described in co-owned,co-pending U.S. application Ser. No. 08/557,289 filed Nov. 14, 1995 andU.S. Pat. No. 5,686,414 and corresponding International Publication Nos.WO 97/17990 (PCT/US96/18632) and WO 97/17989 (PCT/US96/18416), all ofwhich are incorporated herein by reference), ear drops, suppositories,irrigation fluids (for, e.g., irrigation of wounds) or medicatedshampoos. For example, for topical administration in drop form, about 10to 200 μL of a BPI protein product composition may be applied one ormore times per day as determined by the treating physician.

Those skilled in the art can readily optimize effective dosages andadministration regimens for therapeutic compositions comprising BPIprotein product, as determined by good medical practice and the clinicalcondition of the individual patient.

Other aspects and advantages of the present invention will be understoodupon consideration of the following illustrative examples. Example 1addresses the construction of an expression vector, pING1742, encodingrBPI(10-193)C132A. Example 2 addresses transformation of CHO cells withpING1742 and selection of the highest producing clones secretingrBPI(10-193)C132A. Example 3 addresses the production and purificationof rBPI(10-193)C132A in 2-L and 500-L fermenters. Example 4 addressesthe biochemical characterization of rBPI(10-193)C132A and rBPI₂₁.Examples 5, 6 and 7 respectively address the in vitro LPS-bindingactivity in a competition binding assay and in an assay measuring rateof complex formation using rate nephelometry, bactericidal activity, andLPS neutralization activity of rBPI(10-193)C132A as compared to rBPI₂₁.Example 8 addresses the in vivo activity of rBPI(10-193)C132A.

EXAMPLE 1 Construction of Expression Vector pING1742

The rBPI(10-193)C132A expression vector, pING1742, was constructed asfollows. The expression vector pING4155 was first constructed byligating a BamHI-Bsal fragment containing the neo gene from pING3174with a Bsal-XhoI of fragment containing the CMV promoter and rBPI₂₁ genefrom pING4144 (including an optimized Kozak translation initiation siteat residue -27 of the signal) and an XhoI-BamHI fragment containing themouse (kappa) light chain 3' untranslated region from pING4537(pING3174, pING4144 and pING4537 are described in U.S. Pat. No.5,420,019, incorporated by reference). The resulting pING4155 vectorcontains the gene encoding rBPI₂₁ fused to the human IgG enhancer, thehuman CMV promoter, a 27 amino acid BPI leader sequence (amino acidresidues -27 through -1 of SEQ ID NO: 2) and the mouse (kappa) lightchain 3' untranslated region. It also contains the neo gene encodingneomycin phosphotransferase, for selection of transfectants resistant tothe antibiotic Geneticin® (G418).

The vector pING1732 was produced by deleting the 0.7 kbpHindIII--HindIII fragment of pING4155 containing the human Ig enhancer.Then, the 27 nucleotides encoding amino acids 1 through 9 of the matureportion of rBPI₂₁ were deleted from pING1732 by overlap PCR mutagenesisusing the following primers:

Primer 1: 5'-CTGCTCTAAAAGCTGCTGCAG-3' (SEQ ID NO: 3)

Primer 2: 5'-CCAGGCCCTTCTGGGAGGCCGCTGTCACGGCGG-3' (SEQ ID NO: 4)

Primer 3: 5'-GCCGTGACAGCGGCCTCCCAGAAGGGCCTGGAC-3' (SEQ ID NO: 5)

Primer 4: 5'-CTGGGAACTGGGAAGCTG-3' (SEQ ID NO: 6)

Overlapping complementary primers 2 and 3 incorporated the 27 bpdeletion of nucleotides encoding amino acids 1 through 9, while primers1 and 4 encoded nucleotides immediately upstream and downstream,respectively, of unique SalI and EcoRI sites in pING1732. First,fragments were obtained by PCR amplification using the combination ofoligonucleotide primers 1 and 3, and primers 2 and 4. After theseindividual fragments were obtained, they were annealed, extended andre-amplified using primers 1 and 4. This amplified fragment was thendigested with SalI and EcoRI and cloned into SalI-EcoRI-digestedpING1732 to generate the plasmid pING1742.

To confirm that no mutations had occurred during PCR, the SalI-EcoRIregion from pING1742 was sequenced. No changes were observed in themature coding region for BPI. However, a two base-pair change (ACC->GCT)was found in DNA encoding the signal sequence, which resulted in theconversion of a Thr to an Ala at amino acid position -6 relative to thestart of the mature protein sequence.

EXAMPLE 2 Transformation of CHO Cells with pING1742

CHO-K1 cells (American Type Culture Collection (ATCC) Accession No.CCL61) were adapted to growth in serum-free Ex-Cell 301 medium asfollows. CHO-K1 cells grown in Ham's F12 medium were trypsinized,centrifuged and resuspended in Ex-Cell 301 medium. Cells were grown in a125-ml flask at 100 rpm and passaged every two to three days in either a125-ml or 250-ml flask.

These Ex-Cell 301-adapted CHO cells were transfected by electroporationwith pING1742. Prior to transfection, pING1742 was digested with NotI,which linearizes the plasmid. Following a 48-hour recovery, cells wereplated at approximately 10⁴ cells/well into 96-well plates containingEx-Cell 301 medium supplemented with 0.6 mg/mL G418 (Life Technologies,Gaithersburg, Md.). At approximately 2 weeks, supernatants fromapproximately 250 wells containing single colonies were screened byEUTSA for the presence of BPI-reactive protein using an anti-BPImonoclonal antibody.

Fifteen clones having the highest expression levels were transferred to24-well plates containing Ex-Cell 301 medium. To screen forproductivity, the cells were grown in 24-well plates containing Ex-Cellmedium supplemented with 2 % FBS and 40 μL sterile S-Sepharose beads for10 days, after which the beads were removed, washed with low salt buffer(0.1 M NaCl in 10 mM Na acetate, pH 4.0) and the BPI eluted with 1.5 MNaCl in the same buffer. The levels of secreted rBPI(10-193)C132A weredetermined by ELISA. Western blot analysis of eluates run on a 12 %non-reducing SDS gel revealed a prominent band which migrated slightlyfaster than rBPI₂₁.

The top eight producers were transferred to sterile 125 mL Erlenmeyerflasks and grown in Ex-Cell medium. These cells were evaluated again forproductivity by growing them in flasks containing Ex-Cell 301 mediumsupplemented with 2% FBS and 1 % (V/V) sterile S-sepharose beads. TherBPI(10-193)C132A was eluted from the S-Sepharose beads that had beenincorporated in the culture medium and the levels of rBPI(10-193)C132Adetermined by HPLC. Clone 139, which was among the highest producers,was chosen for further growth and product production.

EXAMPLE 3 Production and Purification of rBPI(10-193)C132A

Large quantities of rBPI(10-193)C132A were produced for characterizationby growing Clone 139 cells in 2-liter research fermenters (Biolafitte,St. Germain en Laye, France) and then in a 500 liter ABEC fermenter(ABEC, Allentown, Pa.). Protein product obtained from the 2-literfermenters was used for the in vitro studies described below, whileproduct obtained from the 500 liter fermenter was used for animaltoxicology and efficacy studies.

A. Growth in Two-Liter Fermenters

Clone 139 cells were passaged in spinner flasks of increasing volumescontaining Ex-Cell medium supplemented with 1% FBS until sufficientvolume and cell density was achieved to inoculate the 2 literbioreactors at approximately 2×10⁵ cells/mL. Cells were grown in three2-liter fermenters in Ex-Cell medium supplemented with 1% FBS, at 37 °C., pH 7.2, 150 rpm with dissolved oxygen maintained at 5-10%. Largesterile SP-Sepharose beads (Pharmacia and Upjohn, Piscataway, N.J.) wereadded at 1.5% (V/V). The initial glucose level was approximately 3.5 g/Land glucose was pulsed daily to 3 g/L during the course of the run. Thefermentation was terminated at 238 hours, at which time the cellviabilities were from 63%, 80% and 84%.

Following fermentation, the beads from each fermenter were harvested,allowed to settle, and washed several times with 10 mM Naphosphate/0.15M NaCl, pH 7.0, to remove cellular components and weaklybound impurities from the beads. The washed beads were packed into acolumn, washed with 10 mM Na phosphate, 0.25 M NaCl, pH 7.0, and elutedwith the same buffer containing 0.8 M NaCl, 5 mM glycine. The eluate wasthen diluted with three volumes of sterile water for injection (WFI),loaded onto a CM-spherodex column (Sepracor, Marlborough, Mass.) andwashed with 10 mM Na phosphate, 0.25 M NaCl, pH 7.0, followed by 20 mMNa acetate, 0.2 M NaCl, pH 4.0, followed by 20 mM Na acetate, 0.3 MNaCl, pH 4.0, and sample was eluted at 1.0 M NaCl in the same buffer.Following concentration on a Centricon membrane with a 10,000 MW cutoff(Amicon, Beverly, Mass.), the eluate from the CM column was loaded ontoa Sephacryl S-100 column (Pharmacia and Upjohn) equilibrated with 5 mMNa citrate, 0.15 M NaCl, pH 5.0. Fractions containing rBPI(10-193)C132Aidentified by absorbance at 280 nm were pooled, concentrated on anAmicon filter to 1.9 mg/mL and formulated with 0.002% polysorbate 80 (TBaker, Phillipsburg, N.J.), 0.2% poloxamer 188 (Pluronic F-68, BASF,Parsippany, N.J.). The final preparation was filter sterilized using a0.2 μm filter.

B. Growth in 500-Liter Fermenter

Clone 139 cells were passaged in fetuin-free Ex-Cell medium with 1% FBSin a series of spinner flasks of increasing volumes to provide inoculumfor the 35L Bellco spinner flask (Bellco Glass, Vineland, N.J.), whichin turn provided the inoculum for the 500 liter ABEC fermenter. Cellswere grown in complete Ex-Cell medium without fetuin but supplementedwith 1% FBS, additional glucose (to 10 g/L) and glutamine (to 10 mM).The fermenter was operated in a fed-batch mode with one 0.5% PrimatoneRL supplement pulse and one glucose/glutamine pulse added during therun. Five to six liters of large SP-Sepharose beads were added 24 hoursafter the 500 liter fermenter was inoculated. The pH was controlledmanually with 10% sodium bicarbonate to pH 7.0, oxygen was controlled at5% and temperature at 37° C. Agitation was maintained at 25 rpm with twothree-blade paddle impellers. The fermentation run was terminated at 184hours, at which time the cell viability was 90%.

As described above for the 2-liter fermentation, the beads were allowedto settle following fermentation and then washed several times with lowsalt (0.1M) phosphate buffer. The steps for this purification weresimilar to those described above for the 2-liter samples except that apH 3.0 viral inactivation step was included after elution from theS-Sepharose beads and a second CM-spherodex column was included as aconcentration step. For the second CM column, the eluate was dilutedwith three volumes of WFI, the pH adjusted to 5.0, the column wasequilibrated and washed with 20 mM Na acetate, 0.3 M NaCl, pH 5.0 andthe sample was eluted at 1.0 M NaCl in the same buffer. TherBPI(10-193)C132A was eluted from the Sephacryl S-100 column in 5 mM Nacitrate, 0.15 M NaCl, pH 5.0, adjusted to 2 mg/mL, and filtered througha 0.2 μm filter. The rBPI(10-193)C132A was then formulated with 0.002%polysorbate 80, 0.2% poloxamer 188, sterile filtered, and filled into 10mL Type I glass serum vials.

EXAMPLE 4 Biochemical Characterization of rBPI(10-193)C132A

A. Protein from the 2-Liter Fermentations

The purified rBPI(10-193)C132A product from Example 3 was observed to bea single band that migrated slightly faster on SDS polyacrylamide gelelectrophoresis (SDS-PAGO) than the rBPI₂₁ band, consistent with thedeletion of nine N-terminal amino acids from rBPI₂₁. Sequence analysisdemonstrated that the rBPI(10-193)C132A contained the predictedN-terminal sequence of SQKGLDYASQQGTAALQKEL. On mass spectroscopyanalysis (ESI-MS) two components were observed, one with a mass of20,470 daltons, which was consistent with the predicted mass of 20,472daltons for rBPI(10-193)C132A, and a second with a mass of 20,255daltons, consistent with the predicted mass of 20,258 daltons forrBPI(10-191). The ion-exchange HPLC profiles (Hewlett-Packard, Model1050, Palo Alto, Calif.) of rBPI(10-193)C132A and rBPI₂₁ both exhibitedsingle peaks with similar retention times.

B. Protein from the 500-Liter Fermentation

On SDS-PAGE, the rBPI(10-193)C132A was a single band that migratedslightly faster than the rBPI₂₁ band. On mass spectroscopy, there was amajor component with a mass of 20,471 daltons, which is consistent withthe predicted mass of 20,474 Da for rBPI(10-193)C132A), and two minorcomponents with a mass of 20,668 daltons, which is consistent withaddition of N-Acetylhexosamine (predicted mass 20,677 daltons) and amass of 20,843 daltons, which is consistent with addition ofN-Acetylhexosamine plus hexose (predicted mass 20,839 daltons). Asimilar component with added N-Acetylhexosamine is routinely observedduring production of rBPI₂₁.

On reverse phase HPLC (Shimadzu, Kyoto, Japan) both therBPI(10-193)C132A and rBPI₂₁ eluted as one major peak and one minorpeak. However, the rBPI(10-193)C132A peaks eluted slightly earlier thanthe corresponding rBPI₂₁ peaks in the control. The minor peak in therBPI(10-193)C132A profile most likely represents the glycosylated formsidentified in the mass spectrum. The ion-exchange HPLC profiles ofrBPI(10-193)C132A and rBPI₂₁ both exhibited single peaks with similarretention times.

Tryptic mapping analysis was performed according to conventionalmethods. Acetone precipitated rBPI₂₁ or rBPI(10-193)C132A was firsttreated with dithiothreitol (DTT) followed by iodoacetamide and thenwith trypsin. The trypsin-treated product was analyzed by HPLC (BeckmanModel 126) with a C18 column (Beckman Ultrasphere). In rBPI₂₁, there aretwo N-terminal tryptic fragments (T1 and Ala-T1) which result fromimprecise cleavage of the leader sequence. As predicted, the tryptic mapof the rBPI(10-193)C132A was similar to rBPI₂₁ except that theN-terminal fragments were missing.

EXAMPLE 5 In Vitro LPS-Binding Activity of rBPI(10-193)C132A

A. In a Competition Binding Assay

The ability of purified rBPI(10-193)C132A produced according to Example3A and rBPI₂₁ to compete with labeled rBPI₂₁ for binding to LPS wasevaluated in a competition binding assay. Briefly, a fixed concentration(0.5 nM) of ¹²⁵ -labeled rBPI₂₁ was mixed with unlabeled rBPI₂₁ orrBPI(10-193)C132A at dilutions ranging from 5 μM to 0.01 nM in DMEMcontaining HEPES buffer and bovine serum albumin (BSA) [U.S.Biochemicals, Cleveland, Ohio] and 100 μL of the mixture was added toImmulon-II plate wells pre-coated with 2.5 μg/mL E. coli J5 LPS(Calbiochem, San Diego, Calif.). The plates were incubated at 4° C. for5 hours and washed 3 times with the DMEM medium. 75 μL of 0.1 N NaOH wasadded and the bound ¹²⁵ I-rBPI₂₁ was removed and counted. The resultsdemonstrated that both proteins competed similarly with radiolabeledrBPI₂₁.

B. In an Assay Measuring Rate of Complex Formation

The LPS binding activity of rBPI(10-193)C132A was compared to rBPI₂₁using rate nephelometry. This approach for evaluating rBPI₂₁ binding toLPS measures the rate of increase of light scattering as a result ofLPS-BPI protein product complex formation in solution. All of theexperiments were performed with a Beckman Array 360 Rate Nephelometerwhich automatically mixes samples, measures light scattering andperforms rate calculations.

Prior experiments using this approach examined optimal LPS species andconcentration, assay specificity, assay reproducibility and correlationof assay results to bactericidal assays. It was observed that E. coli J5LPS and lipid A formed complexes with rBPI₂₁ that could be measured inthe nephelometer, but E. coli O111:B4 LPS did not form measurablecomplexes. Based on results of these studies, E. coli J5 LPS was chosenfor use at a concentration (in the flow cell) of 49.4 to 61.7 μg/ml,depending on the LPS lot, in combination with rBPI₂₁ concentrations (inthe flow cell) from 5 to 30 μg/ml. The optimal rBPI₂₁ concentrationrange, which must be determined for each LPS lot, was from about 15 to25 μg/ml which represented the most linear portion of the curve. Theoptimal range for the aggregation rate RT) values was from 700 to 2000.Lower concentrations of rBPI₂₁ were needed to achieve the sameaggregation rate values when the formulation buffer was changed toinclude PLURONIC P103 or when the NaCl concentration was increased. Theaddition of either recombinant lipopolysaccharide binding protein(rLBP₅₀) which binds to LPS, or heparin which binds to BPI proteinproducts, inhibited the formation of rBPI₂₁ -LPS aggregates,demonstrating the specificity of the interaction. Assay reproducibilitywas confirmed by testing multiple lots of BPI and testing the same lotof rBPI₂₁ multiple times. Nephelometric analysis of rBPI₂₁ samples thathad been partially inactivated by treatment at 45 ° C. for one weekcorrelated well with those from broth microdilution bactericidal assayswith E. coli J5 cells.

Nephelometry experiments comparing rBPI(10-193)C132A and rBPI₂₁ werecarried out as follows. Sonicated LPS [E. coli J5 LPS Lot No. 30119Bfrom List Biochemicals] and either rBPI(10-193)C132A or rBPI₂₁ [both ofwhich were formulated in 0.2% PLURONIC F68 (poloxamer 188), 0.002% TWEEN80 (polysorbate 80), 5 mM citrate, pH 5.0, 150 mM NaCl] were diluteddirectly into a PBS buffer (supplemented with PEG) supplied by Beckman.The LPS concentration was fixed while the BPI protein productconcentration varied within each experiment. Two concentrations of LPSwere tested: 24.7 and 49.4 μg/ml LPS. Each reaction was initiated byaddition of 600 μl of the PBS-PEG buffer to the flow cell followed by 42μl of the BPI protein product dilution. After a baseline wasestablished, 42 μl of the E. coli J5 LPS solution was added. Afteraddition of the last component, the nephelometer measures the rate ofcomplex formation based on the extent of light scatter. The data wereanalyzed by dividing the RT values for each test sample containing agiven BPI protein product concentrations by the corresponding RT valuesfor the standard to generate a percent of control value. For each BPIprotein product concentration tested, the maximum aggregation rate wasdetermined and a curve generated. Only points to the left of the maximumvalue (point of equivalence) were used for comparative analysis ofvarious BPI protein product samples. The relative activity of samplescan be measured by comparing the RT values for test and standard lots inthe linear region of the curves. Either a point to point or curve fitapproach can be used.

In addition to testing purified rBPI(10-193)C132A and purified rBPI₂₁[which contains about 7.8% rBPI(10-193)C132A], an equal mixture of theseproteins as well as a rBPI₂₁ preparation with 16% rBPI(10-193)C132A wasevaluated (at 49.4 μg/ml LPS only). The results demonstrated that at49.4 μg/ml LPS, rBPI(10-193)C132A achieved aggregation rates similar tothat of rBPI₂₁ at an approximately 25% lower concentration. TherBPI(10-193)C132A also achieved a higher maximum aggregation rate thanthat of rBPI₂₁ at both 24.7 and 49.4 μg/ml LPS. An equal mix of the twomolecules yielded a curve that ran between rBPI₂₁ and rBPI(10-193) whilethe rBPI₂₁ lots with 7.8% and 16% 10-193 behaved in an identical mannerto each other. A point to point analysis of the results (LPS at 49.4μg/ml) revealed that the rBPI(10-193) was approximately twice as potentas rBPI₂₁ in this assay.

EXAMPLE 6 In Vitro Bactericidal Activity of rBPI(10-193)C132A

All of the assays in this example were conducted with rBPI(10-193)C132Aproduced in the 2-liter fermenters according to Example 3A.

A. Effect on E. coli in a Radial Diffusion Assay

This radial diffusion assay compared the bactericidal effect of purifiedrBPI(10-193)C132A and rBPI₂₁ on E. coli J5, which is aUDP-galactose-4-epimerase "rough" mutant of the smooth strain E. coli011B4, and is relatively sensitive to rBPI₂₁. E. coli J5 cells (Mannionet al., J. Clin. Invest., 85:853-860 (1990); List BiologicalLaboratories, Campbell, Calif.) were grown to exponential phase,centrifuged and washed twice in 10 mM Na phosphate, pH 7.4, and added ata final concentration of approximately 1×10⁶ CFU/ml to molten agarosesupplemented with 3% Trypticase Soy Broth (TSB, DIFCO Laboratories,Detroit, Mich.), 10 mM Na phosphate. Wells of 3 mm diameter wereprepared in the hardened agarose and 5 μL of serially diluted rBPI₂₁ orrBPI(10-193)C132A was added to the wells. The plates were incubated at37° C. for 3 hours to allow diffusion to occur, and then a moltenagarose overlay containing 6% TSB was added. The plates were incubatedovernight at 37° C. and the net area of inhibition was plotted vs.concentration. The results demonstrated that rBPI(10-193)C132A andrBPI₂₁ behaved in a similar manner in this assay.

B. Effect on S. Aureus L-Form in a Radial Diffusion Assay

This radial diffusion assay compared the bactericidal effect of purifiedrBPI(10-193)C132A and rBPI₂₁ on the gram-positive bacteria S. aureusgrown as L-forms without their cell walls. As described in U.S. Pat. No.5,578,572, incorporated herein by reference, S. aureus L-form cells weregrown to log phase in heart infusion (HI) broth supplemented with 3.5%NaCl, 10 mM CaCl₂ and 1000 U/mL penicillin G. The cells were diluted toapproximately either 5×10⁴ or 5×10⁵ cells/mL in molten 0.8% agarosecontaining the NaCl-supplemented HI medium, and 8 ml of the cell-agarosesuspension was poured into 10 cm plates. Wells of 3 mm diameter wereprepared, and 5 μL of serially diluted rBPI₂₁ or rBPI(10-193)C132A wasadded to the wells. The plates were incubated at 37° C for 24 hours andthe net area of inhibition was plotted vs. concentration. The resultsdemonstrated that both rBPI₂₁ and rBPI(10-193)C132A inhibited growth ofthe S. aureus L-forms, at cell densities of about 5×10⁴ and 5×10⁵, in asimilar fashion in this assay.

C. Effect on E. coli J5 in a Broth Microdilution Assay

This broth microdilution assay compared the bactericidal effect ofpurified rBPI(10-193)C132A and rBPI₂₁ on E. coli J5. E. coli J5 cellswere grown overnight in tryptone yeast extract (TYE) broth and then tologarithmic phase in TEA medium as previously described in Horwitz etal., Infect. Immun., 63:522-527 (1995). The cells were inoculated atapproximately 1×10⁴ and 1×10⁵ cells/ML in heart infusion (HI) broth, and95 μL was added to 96 well plates. Five μL of various dilutions ofrBPI(10-193)C132A or rBPI₂₁, prepared in formulation buffer, was addedto each well and the plates were incubated at 37° C. for 24 hours. Theresults demonstrated that rBPI(10-193)C132A and rBPI₂₁ have similaractivities in these assays.

EXAMPLE 7 In Vitro LPS Neutralization Activity of rBPI(10-193)C132A

The assay in section A of this example was conducted withrBPI(10-193)C132A produced in the 2-liter fermenters according toExample 3A, while the assay in section B of this example was conductedwith rBPI(10-193)C132A produced in the 500-liter fermenter according toExample 3B.

A. Activity in a RAW Cell Proliferation Assay

The RAW cell proliferation assay was used to compare the in vitro LPSneutralization activity of rBPI₂₁ and rBPI(10-193)C132A. In this assay,the LPS inhibits the proliferation of RAW cells, and rBPI₂₁ neutralizesthis effect of LPS.

Mouse RAW 264.7 cells (ATCC Accession No. T1B71), maintained in RPMI1640 medium (GIBCO), supplemented with 10 mM HEPES buffer (pH 7.4), 2 mML-glutamine, penicillin (100 U/mL), streptomycin (100 /μg/mL), 0.075%sodium bicarbonate, 0.15M 2-mercaptoethanol and 10% fetal bovine serum(Hyclone, Inc., Logan, Utah), were first induced by incubation in thepresence of 50 U/mL recombinant mouse γ-interferon (Genzyme, Cambridge,Mass.) for 24 hours prior to assay. Induced cells were then mechanicallycollected and centrifuged at 500×g at 4° C. and then resuspended in 50mL RPMI 1640 medium (without supplements), re-centrifuged and againresuspended in RPMI 1640 medium (without supplements). The cells werecounted, their concentration adjusted to 2×10⁵ cells/mL and 100 μLaliquots were added to each well of a 96-well plate.

The cells were then incubated for about 15 hours with E. coli O113 LPS(Control Standard, Assoc. of Cape Code, Woods Hole, Mass.), which wasadded in 100 μL/well aliquots at a concentration of 1 ng/mL inserum-free RPMI 1640 medium (this concentration being the result oftitration experiments in which LPS concentration was varied between 50pg/mL and 100 ng/mL). This incubation was performed in the absence orpresence of rBPI₂₁ or rBPI(10-193)C132A in varying concentrationsbetween 25 ng/mL and 50 μg/mL. Recombinant human rBPI₂₁, also designatedrBPI₂₁ Δcys, which is rBPI 1-193 with alanine substituted at position132 for cysteine [see co-owned U.S. Pat. No. 5,420,019], was used as apositive control at a concentration of 1 μg/mL. Cell proliferation wasquantitatively measured by the addition of 1 μCi/well [³ ]-thymidine 5hours after the time of initiation of the assay. After the 15-hourincubation, labeled cells were harvested onto glass fiber filters with acell harvester (Inotech Biosystems, INB-384, Sample Processing andFilter Counting System, Lansing, Mich.). The LPS-mediated inhibition ofRAW 264.7 cell proliferation is dependent on the presence of LBP, asadded to the reaction mixture either as a component of serum or asrecombinant LBP (at a concentration of 1 μg/mL.

In these experiments, both rBPI₂₁ and rBPI(10-193)C132A similarlyinhibited the LPS-mediated inhibition of RAW cell proliferation.

B. Activity in a TNF Inhibition Assay

A tumor necrosis factor (TNF) inhibition assay was used to compare thein vitro LPS neutralization activity of rBPI₂₁ and therBPI(10-193)C132A. In this assay, the LPS, in combination with purifiedLBP (or serum containing LBP) stimulates synthesis of TNF by THP-1 cells(a human monocyte cell line), and rBPI₂₁ neutralizes this effect of LPS.

THP.1 cells (ATCC Accession No. TIB-202) were maintained in RPMI(GibcoBRL, Gaithersburg, Md.) with 10% FBS and were cultured in RPMIwith 10% FBS plus 50 ng/ml 1,25 dihydroxy vitamin D (BIOMOL ResearchLaboratories Inc. Plymouth Meeting, Pa.) for three days prior totreatment with LPS to induce CD14 expression. Before inducing with LPS,cells were washed three times with RPMI and suspended in either RPMIwith 10% FBS or in serum free medium [RPMI supplemented with 1% HB101(Irvine Scientific, Santa Ana, Calif.)]. Expression of TNF was inducedwith 1 ng/ml E. coli 0128 LPS (Sigma, St. Louis, Mo.) in 96 well plateswith approximately 5×10⁴ cells per well. Plates were incubated for threehours at 37° C., 5% CO₂, then an aliquot of the supernatant liquid wasremoved and assayed for TNF by the WEHI 164 toxicity assay, usingCellTiter 96™ AQ (Promega Corp., Madison, Wis.) to monitor cellviability. Recombinant human TNFα (Genzyme Diagnostics, Cambridge,Mass.) was used as a positive standard. Both rBPI₂₁ andrBPI(10-193)C132A similarly inhibited LPS-induced stimulation of TNFsynthesis.

EXAMPLE 8 In Vivo Biological Activity of rBPI(10-193)C132A

The in vivo assays described below were performed using the purifiedrBPI(10-193)C132A produced in the 500-liter fermenter according toExample 3B.

A. Toxicity Study in Rats

Toxicity profiles of rBPI₂₁ and rBPI(10-193)C132A were compared in rats.In this study, groups of six male and six female Sprague-Dawley ratsreceived either vehicle control (formulation buffer), low (50 mg/kg/day)or high (120 mg/kg/day) doses of either rBPI₂₁ or rBPI(10-193)C132A.Doses were administered by continuous intravenous infusion via anindwelling femoral catheter for three consecutive days at an infusionrate of 4.2 mL/kg/hour (100 mL/kg/day). Clinical observations wererecorded at least twice daily and body weights were recorded daily.Blood and urine samples were collected near termination for hematology,clinical chemistry and urinalysis assessments. At termination, organswere weighed and tissues collected by histopathological examination.There were no deaths or significant test article-related effects. Thedata indicated similar toxicity profiles for rBPI₂₁ and therBPI(10-193)C132A when given by continuous infusion.

B. Pharmacokinetics

The pharmacokinetics of rBPI₂₁ and rBPI(10-193)C132A at 2 mg/kg wereinvestigated in rats. The plasma clearances of rBPI₂₁ andrBPI(10-193)C132A were well described by a tri-exponentialpharmacokinetic disposition function. No statistical differences in thepharmacokinetic parameters among the rBPI products were determined (nonparametric Wicoxon rank test, p<0.05). Most of the administered drug(>96%) was cleared with an alpha phase half-life of 0.2-0.4 minutes anda beta half-life of 3.9-4.3 minutes, while the remainder was clearedduring the gamma phase with a half-life of 27-33 minutes. The volume ofdistribution of the central compartment (Vc) was 41-45 mL/kg, and theclearance rate (CL) was 24-30 mL/min/kg. The steady state volume ofdistribution was 152-184 mL/kg.

C. Efficacy in Mouse Endotoxin Challenge

Two separate studies were conducted to examine relative potencies ofrBPI₂₁ and rBPI(10-193)C132A in a mouse model of lethal endotoxemiagenerally according to Ammons et al., in "Novel Therapeutic Strategiesin the Treatment of Sepsis," Morrison and Ryan, eds., Marcel Dekker, NewYork (1996), pages 55-69. In both studies, there were 14 mice in eachtreatment and control group. In the first study, CD1 mice werechallenged intravenously with 25 mg/kg of lipopolysaccharide (LPS) fromE. coli O111:B4. Immediately after the challenge, the mice were treatedintravenously with rBPI₂₁ or rBPI(10-193)C132A at doses of 15, 20, 25and 30 mg/kg, or with the control vehicle (formulation buffer only).Mortality was recorded twice daily for seven days.

The results from the first study, shown in Table 1 below, indicate thattreatment with both rBPI(10-193)C132A and rBPI₂₁ significantly increasedsurvival compared to the vehicle controls. In addition,rBPI(10-193)C132A was at least two-fold more potent than rBPI₂₁ with asimilar survival benefit seen with a two-fold lower dose ofrBPI(10-193)C132A compared to rBPI₂₁.

                  TABLE 1                                                         ______________________________________                                                  No. of Survivors out of 15                                          Dose (mg/kg)                                                                              Control  rBPI.sub.21                                                                            rBPI(10-193)C132A                               ______________________________________                                        0(Vehicle)  0        NA.sup.1 NA                                                15  0 15**.##                                                                 20  4 15**,#                                                                  25  11** 15**                                                                 30  13** 13**                                                               ______________________________________                                         .sup.1 NA, Not Applicable                                                     **, p < 0.01 vs. control                                                      #, p < 0.05 vs. rBPI.sub.21                                                   ##, p < 0.01 vs. control                                                 

In the second study, a wider range of rBPI(10-193)C132A doses (5, 10,15, 20, 25, 30 mg/kg) was studied. The results, shown in Table 2 below,confirm that while both rBPI₂₁ and rBPI(10-193)C132A offered asignificant survival benefit over the control, as in the first study,rBPI(10-193)C132A was at least two-fold more potent, achieving similarefficacy as rBPI₂₁ with a 2-fold lower dose.

                  TABLE 2                                                         ______________________________________                                                  No. of Survivors out of 15                                          Dose (mg/kg)                                                                              Control  rBPI.sub.21                                                                            rBPI(10-193)C132A                               ______________________________________                                        0(Vehicle)  2        NA.sup.1 NA                                                5  ND.sup.1 3                                                                 10  ND 10*                                                                    15  ND 14**                                                                   20  7 14**,#                                                                  25  13** 15**                                                                 30  14** 15*                                                                ______________________________________                                         .sup.1 NA, Not Applicable; ND, Not Done                                       **, p < 0.05 vs. control                                                      **, p < 0.01 vs. control                                                      #, p < 0.05 vs. rBPI.sub.21                                              

D. Efficacy In Murine Model Of Lethal Bacteremia

Two separate studies were conducted to examine the relative potencies orrBPI₂₁ and rBPI(10-193)C132A in a mouse model of lethal bacteremia. Inboth studies, there were 20 mice per treatment group. In the firststudy, CD1 mice were challenged with 6.8×10⁷ colony forming units (CFU)of E. coli 07:K1 administered intravenously. Immediately after thechallenge, the mice were treated intravenously with rBPI₂₁ orrBPI(10-193)C132A at doses of 10, 20 and 30 mg/kg, or with controlvehicle (formulation buffer only). Mortality was recorded twice dailyfor seven days.

The results from the first study, shown in Table 3 below, demonstrate asignificant increase in survival for the groups treated with 10 and 30mg/kg of rBPI₂₁ (p<0.05 vs. control). While a similar significantincrease in survival was not observed with the rBPI(10-193)C132A vs.control, there was not a significant difference in survival advantagebetween the rBPI₂₁ and rBPI(10-193)C132A -treated groups in this study.

                  TABLE 3                                                         ______________________________________                                                  No. of Survivors out of 20                                          Dose (mg/kg)                                                                              Control  rBPI.sub.21                                                                            rBPI(10-193)C132A                               ______________________________________                                        0(Vehicle)  6                 NA                                                10  14* 12                                                                    20  12 10                                                                     30  14* 10                                                                  ______________________________________                                         *, p < 0.05 vs. control                                                  

To more fully characterize the effects of rBPI₂₁ and rBPI(10-193)C132Ain this model, a second study was conducted in which a wider range ofdoses was studied. In this study, CD1 mice were challenged with 2.57×10⁸colony forming units (CFU) of E. coli O7:K1 administered intravenously.Immediately after the challenge, the mice were treated intravenouslywith 1.0, 3.0, 10 and 30 mg/kg rBPI₂₁ and 0.3, 1.0, 3.0, 10 and 30 mg/kgrBPI(10-193)C132A. The results, shown in Table 4 below, indicate thatboth proteins provided protection, and that there was no significantdifference in the protective effects of the two variants at any dose.

                  TABLE 4                                                         ______________________________________                                                  No. of Survivors out of 20                                          Dose (mg/kg)                                                                              Control  rBPI.sub.21                                                                            rBPI(10-193)C132A                               ______________________________________                                          0(Vehicle) 6                                                                0.3                  ND.sup.1 6                                                 1.0  4 6                                                                      3.0  9 10*                                                                    10  13** 8                                                                    30  11* 14**                                                                ______________________________________                                         .sup.1 ND, Not Done                                                           *, p < 0.05 vs. control                                                       **, p < 0.01 vs. control                                                 

E. Cardiovascular Effects in Conscious Rats

A series of experiments were conducted to determine the relative effectsof rBPI₂₁ and rBPI(10-193)C132A on blood pressure in rats. Each rat wasanesthetized with a mixture of ketamine (Fort Dodge Labs, Fort Dodge,Id.) and Rompum (Bayer Corp., Shawnee Mission, Kans.). A catheter wasthen placed in the right carotid artery and connected to a pressuretransducer to record blood pressure. A second catheter was placed in theright jugular vein to inject rBPI or vehicle. The rats were then allowedto recover before the experiments began. Experiments were initiated whenthe rats were alert, mobile and when blood pressure was stable withinthe normal range. rBPI₂₁, rBPI(10-193)C132A or control vehicle(formulation buffer) were then injected as a bolus over 15 seconds andmean arterial blood pressure (mm Hg) was recorded over the next 60minutes.

In preliminary experiments, it was determined that doses of 20 and 30mg/kg of rBPI₂₁ had no significant effect on blood pressure relative tothe vehicle but that a dose of 40 mg/kg resulted in a significantdecrease in blood pressure that was evident within 5 minutes. Thishypotensive response was greatest 15 minutes after the injection whenblood pressure had decreased by 48±12 mm Hg (mean ±SE; p>0.05). After 60minutes, the blood pressure of the rBPI₂₁ -treated animals recovered andwas not significantly different from that of the vehicle-treatedanimals.

To compare effects of rBPI₂₁ and rBPI(10-193)C132A, groups of 5 ratswere given 40 mg/kg of each drug substance or vehicle control, and bloodpressure responses were analyzed as area under the curve (AUC). FIG. 1shows that, as previously observed, rBPI₂₁ caused a significant drop inblood pressure indicated by the elevated AUC relative to the vehiclecontrol. By comparison, rBPI(10-193)C132A had no significant effect onblood pressure compared with the vehicle control. A dose of 50 mg/kgrBPI₂₁ (N=4 rats) had an even greater hypotensive effect than that ofthe 40 mg/kg dose as indicated by a further increase in the AUC inFIG. 1. At this higher dose, some reduction in blood pressure alsooccurred in rats administered rBPI(10-193)C132A (N=3), but this effectwas not significant compared to the vehicle control.

Numerous modifications and variations of the above-described inventionare expected to occur to those of skill in the art. Accordingly, onlysuch limitations as appear in the appended claims should be placedthereon.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - <160> NUMBER OF SEQ ID NOS: 6                                        - - <210> SEQ ID NO 1                                                        <211> LENGTH: 1813                                                            <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: CDS                                                           <222> LOCATION: (31)..(1491)                                                  <220> FEATURE:                                                                <221> NAME/KEY: mat.sub.-- peptide                                            <222> LOCATION: (124)..(1491)                                                 <220> FEATURE:                                                                <223> OTHER INFORMATION: rBPI                                                  - - <400> SEQUENCE: 1                                                         - - caggccttga ggttttggca gctctggagg atg aga gag aac at - #g gcc agg       ggc      54                                                                                       - #               Met Arg - #Glu Asn Met Ala Arg Gly                         - #                  - # -30                - # -25          - - cct tgc aac gcg ccg aga tgg gtg tcc ctg at - #g gtg ctc gtc gcc ata          102                                                                       Pro Cys Asn Ala Pro Arg Trp Val Ser Leu Me - #t Val Leu Val Ala Ile                       -20      - #           -15      - #           -10                  - - ggc acc gcc gtg aca gcg gcc gtc aac cct gg - #c gtc gtg gtc agg atc          150                                                                       Gly Thr Ala Val Thr Ala Ala Val Asn Pro Gl - #y Val Val Val Arg Ile                    -5         - #     -1   1         - #      5                          - - tcc cag aag ggc ctg gac tac gcc agc cag ca - #g ggg acg gcc gct ctg          198                                                                       Ser Gln Lys Gly Leu Asp Tyr Ala Ser Gln Gl - #n Gly Thr Ala Ala Leu            10                 - # 15                 - # 20                 - # 25       - - cag aag gag ctg aag agg atc aag att cct ga - #c tac tca gac agc ttt          246                                                                       Gln Lys Glu Leu Lys Arg Ile Lys Ile Pro As - #p Tyr Ser Asp Ser Phe                            30 - #                 35 - #                 40              - - aag atc aag cat ctt ggg aag ggg cat tat ag - #c ttc tac agc atg gac          294                                                                       Lys Ile Lys His Leu Gly Lys Gly His Tyr Se - #r Phe Tyr Ser Met Asp                        45     - #             50     - #             55                  - - atc cgt gaa ttc cag ctt ccc agt tcc cag at - #a agc atg gtg ccc aat          342                                                                       Ile Arg Glu Phe Gln Leu Pro Ser Ser Gln Il - #e Ser Met Val Pro Asn                    60         - #         65         - #         70                      - - gtg ggc ctt aag ttc tcc atc agc aac gcc aa - #t atc aag atc agc ggg          390                                                                       Val Gly Leu Lys Phe Ser Ile Ser Asn Ala As - #n Ile Lys Ile Ser Gly                75             - #     80             - #     85                          - - aaa tgg aag gca caa aag aga ttc tta aaa at - #g agc ggc aat ttt gac          438                                                                       Lys Trp Lys Ala Gln Lys Arg Phe Leu Lys Me - #t Ser Gly Asn Phe Asp            90                 - # 95                 - #100                 - #105       - - ctg agc ata gaa ggc atg tcc att tcg gct ga - #t ctg aag ctg ggc agt          486                                                                       Leu Ser Ile Glu Gly Met Ser Ile Ser Ala As - #p Leu Lys Leu Gly Ser                           110  - #               115  - #               120              - - aac ccc acg tca ggc aag ccc acc atc acc tg - #c tcc agc tgc agc agc          534                                                                       Asn Pro Thr Ser Gly Lys Pro Thr Ile Thr Cy - #s Ser Ser Cys Ser Ser                       125      - #           130      - #           135                  - - cac atc aac agt gtc cac gtg cac atc tca aa - #g agc aaa gtc ggg tgg          582                                                                       His Ile Asn Ser Val His Val His Ile Ser Ly - #s Ser Lys Val Gly Trp                   140          - #       145          - #       150                      - - ctg atc caa ctc ttc cac aaa aaa att gag tc - #t gcg ctt cga aac aag          630                                                                       Leu Ile Gln Leu Phe His Lys Lys Ile Glu Se - #r Ala Leu Arg Asn Lys               155              - #   160              - #   165                          - - atg aac agc cag gtc tgc gag aaa gtg acc aa - #t tct gta tcc tcc aag          678                                                                       Met Asn Ser Gln Val Cys Glu Lys Val Thr As - #n Ser Val Ser Ser Lys           170                 1 - #75                 1 - #80                 1 -      #85                                                                              - - ctg caa cct tat ttc cag act ctg cca gta at - #g acc aaa ata gat        tct      726                                                                    Leu Gln Pro Tyr Phe Gln Thr Leu Pro Val Me - #t Thr Lys Ile Asp Ser                          190  - #               195  - #               200              - - gtg gct gga atc aac tat ggt ctg gtg gca cc - #t cca gca acc acg gct          774                                                                       Val Ala Gly Ile Asn Tyr Gly Leu Val Ala Pr - #o Pro Ala Thr Thr Ala                       205      - #           210      - #           215                  - - gag acc ctg gat gta cag atg aag ggg gag tt - #t tac agt gag aac cac          822                                                                       Glu Thr Leu Asp Val Gln Met Lys Gly Glu Ph - #e Tyr Ser Glu Asn His                   220          - #       225          - #       230                      - - cac aat cca cct ccc ttt gct cca cca gtg at - #g gag ttt ccc gct gcc          870                                                                       His Asn Pro Pro Pro Phe Ala Pro Pro Val Me - #t Glu Phe Pro Ala Ala               235              - #   240              - #   245                          - - cat gac cgc atg gta tac ctg ggc ctc tca ga - #c tac ttc ttc aac aca          918                                                                       His Asp Arg Met Val Tyr Leu Gly Leu Ser As - #p Tyr Phe Phe Asn Thr           250                 2 - #55                 2 - #60                 2 -      #65                                                                              - - gcc ggg ctt gta tac caa gag gct ggg gtc tt - #g aag atg acc ctt        aga      966                                                                    Ala Gly Leu Val Tyr Gln Glu Ala Gly Val Le - #u Lys Met Thr Leu Arg                          270  - #               275  - #               280              - - gat gac atg att cca aag gag tcc aaa ttt cg - #a ctg aca acc aag ttc         1014                                                                       Asp Asp Met Ile Pro Lys Glu Ser Lys Phe Ar - #g Leu Thr Thr Lys Phe                       285      - #           290      - #           295                  - - ttt gga acc ttc cta cct gag gtg gcc aag aa - #g ttt ccc aac atg aag         1062                                                                       Phe Gly Thr Phe Leu Pro Glu Val Ala Lys Ly - #s Phe Pro Asn Met Lys                   300          - #       305          - #       310                      - - ata cag atc cat gtc tca gcc tcc acc ccg cc - #a cac ctg tct gtg cag         1110                                                                       Ile Gln Ile His Val Ser Ala Ser Thr Pro Pr - #o His Leu Ser Val Gln               315              - #   320              - #   325                          - - ccc acc ggc ctt acc ttc tac cct gcc gtg ga - #t gtc cag gcc ttt gcc         1158                                                                       Pro Thr Gly Leu Thr Phe Tyr Pro Ala Val As - #p Val Gln Ala Phe Ala           330                 3 - #35                 3 - #40                 3 -      #45                                                                              - - gtc ctc ccc aac tcc tcc ctg gct tcc ctc tt - #c ctg att ggc atg        cac     1206                                                                    Val Leu Pro Asn Ser Ser Leu Ala Ser Leu Ph - #e Leu Ile Gly Met His                          350  - #               355  - #               360              - - aca act ggt tcc atg gag gtc agc gcc gag tc - #c aac agg ctt gtt gga         1254                                                                       Thr Thr Gly Ser Met Glu Val Ser Ala Glu Se - #r Asn Arg Leu Val Gly                       365      - #           370      - #           375                  - - gag ctc aag ctg gat agg ctg ctc ctg gaa ct - #g aag cac tca aat att         1302                                                                       Glu Leu Lys Leu Asp Arg Leu Leu Leu Glu Le - #u Lys His Ser Asn Ile                   380          - #       385          - #       390                      - - ggc ccc ttc ccg gtt gaa ttg ctg cag gat at - #c atg aac tac att gta         1350                                                                       Gly Pro Phe Pro Val Glu Leu Leu Gln Asp Il - #e Met Asn Tyr Ile Val               395              - #   400              - #   405                          - - ccc att ctt gtg ctg ccc agg gtt aac gag aa - #a cta cag aaa ggc ttc         1398                                                                       Pro Ile Leu Val Leu Pro Arg Val Asn Glu Ly - #s Leu Gln Lys Gly Phe           410                 4 - #15                 4 - #20                 4 -      #25                                                                              - - cct ctc ccg acg ccg gcc aga gtc cag ctc ta - #c aac gta gtg ctt        cag     1446                                                                    Pro Leu Pro Thr Pro Ala Arg Val Gln Leu Ty - #r Asn Val Val Leu Gln                          430  - #               435  - #               440              - - cct cac cag aac ttc ctg ctg ttc ggt gca ga - #c gtt gtc tat aaa             1491                                                                       Pro His Gln Asn Phe Leu Leu Phe Gly Ala As - #p Val Val Tyr Lys                           445      - #           450      - #           455                  - - tgaaggcacc aggggtgccg ggggctgtca gccgcacctg ttcctgatgg gc -             #tgtggggc   1551                                                                 - - accggctgcc tttccccagg gaatcctctc cagatcttaa ccaagagccc ct -            #tgcaaact   1611                                                                 - - tcttcgactc agattcagaa atgatctaaa cacgaggaaa cattattcat tg -            #gaaaagtg   1671                                                                 - - catggtgtgt attttaggga ttatgagctt ctttcaaggg ctaaggctgc ag -            #agatattt   1731                                                                 - - cctccaggaa tcgtgtttca attgtaacca agaaatttcc atttgtgctt ca -            #tgaaaaaa   1791                                                                 - - aacttctggt ttttttcatg tg           - #                  - #                   1813                                                                     - -  - - <210> SEQ ID NO 2                                                   <211> LENGTH: 487                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 2                                                         - - Met Arg Glu Asn Met Ala Arg Gly Pro Cys As - #n Ala Pro Arg Trp Val          -30              - #   -25              - #   -20                          - - Ser Leu Met Val Leu Val Ala Ile Gly Thr Al - #a Val Thr Ala Ala Val     15                 - - #10                  - #-5              -1   - #1        - - Asn Pro Gly Val Val Val Arg Ile Ser Gln Ly - #s Gly Leu Asp Tyr Ala                    5    - #              10    - #              15                  - - Ser Gln Gln Gly Thr Ala Ala Leu Gln Lys Gl - #u Leu Lys Arg Ile Lys               20         - #         25         - #         30                      - - Ile Pro Asp Tyr Ser Asp Ser Phe Lys Ile Ly - #s His Leu Gly Lys Gly           35             - #     40             - #     45                          - - His Tyr Ser Phe Tyr Ser Met Asp Ile Arg Gl - #u Phe Gln Leu Pro Ser       50                 - # 55                 - # 60                 - # 65       - - Ser Gln Ile Ser Met Val Pro Asn Val Gly Le - #u Lys Phe Ser Ile Ser                       70 - #                 75 - #                 80              - - Asn Ala Asn Ile Lys Ile Ser Gly Lys Trp Ly - #s Ala Gln Lys Arg Phe                   85     - #             90     - #             95                  - - Leu Lys Met Ser Gly Asn Phe Asp Leu Ser Il - #e Glu Gly Met Ser Ile              100          - #       105          - #       110                      - - Ser Ala Asp Leu Lys Leu Gly Ser Asn Pro Th - #r Ser Gly Lys Pro Thr          115              - #   120              - #   125                          - - Ile Thr Cys Ser Ser Cys Ser Ser His Ile As - #n Ser Val His Val His      130                 1 - #35                 1 - #40                 1 -      #45                                                                              - - Ile Ser Lys Ser Lys Val Gly Trp Leu Ile Gl - #n Leu Phe His Lys        Lys                                                                                             150  - #               155  - #               160             - - Ile Glu Ser Ala Leu Arg Asn Lys Met Asn Se - #r Gln Val Cys Glu Lys                  165      - #           170      - #           175                  - - Val Thr Asn Ser Val Ser Ser Lys Leu Gln Pr - #o Tyr Phe Gln Thr Leu              180          - #       185          - #       190                      - - Pro Val Met Thr Lys Ile Asp Ser Val Ala Gl - #y Ile Asn Tyr Gly Leu          195              - #   200              - #   205                          - - Val Ala Pro Pro Ala Thr Thr Ala Glu Thr Le - #u Asp Val Gln Met Lys      210                 2 - #15                 2 - #20                 2 -      #25                                                                              - - Gly Glu Phe Tyr Ser Glu Asn His His Asn Pr - #o Pro Pro Phe Ala        Pro                                                                                             230  - #               235  - #               240             - - Pro Val Met Glu Phe Pro Ala Ala His Asp Ar - #g Met Val Tyr Leu Gly                  245      - #           250      - #           255                  - - Leu Ser Asp Tyr Phe Phe Asn Thr Ala Gly Le - #u Val Tyr Gln Glu Ala              260          - #       265          - #       270                      - - Gly Val Leu Lys Met Thr Leu Arg Asp Asp Me - #t Ile Pro Lys Glu Ser          275              - #   280              - #   285                          - - Lys Phe Arg Leu Thr Thr Lys Phe Phe Gly Th - #r Phe Leu Pro Glu Val      290                 2 - #95                 3 - #00                 3 -      #05                                                                              - - Ala Lys Lys Phe Pro Asn Met Lys Ile Gln Il - #e His Val Ser Ala        Ser                                                                                             310  - #               315  - #               320             - - Thr Pro Pro His Leu Ser Val Gln Pro Thr Gl - #y Leu Thr Phe Tyr Pro                  325      - #           330      - #           335                  - - Ala Val Asp Val Gln Ala Phe Ala Val Leu Pr - #o Asn Ser Ser Leu Ala              340          - #       345          - #       350                      - - Ser Leu Phe Leu Ile Gly Met His Thr Thr Gl - #y Ser Met Glu Val Ser          355              - #   360              - #   365                          - - Ala Glu Ser Asn Arg Leu Val Gly Glu Leu Ly - #s Leu Asp Arg Leu Leu      370                 3 - #75                 3 - #80                 3 -      #85                                                                              - - Leu Glu Leu Lys His Ser Asn Ile Gly Pro Ph - #e Pro Val Glu Leu        Leu                                                                                             390  - #               395  - #               400             - - Gln Asp Ile Met Asn Tyr Ile Val Pro Ile Le - #u Val Leu Pro Arg Val                  405      - #           410      - #           415                  - - Asn Glu Lys Leu Gln Lys Gly Phe Pro Leu Pr - #o Thr Pro Ala Arg Val              420          - #       425          - #       430                      - - Gln Leu Tyr Asn Val Val Leu Gln Pro His Gl - #n Asn Phe Leu Leu Phe          435              - #   440              - #   445                          - - Gly Ala Asp Val Val Tyr Lys                                              450                 4 - #55                                                    - -  - - <210> SEQ ID NO 3                                                   <211> LENGTH: 21                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: primer         - - <400> SEQUENCE: 3                                                         - - ctgctctaaa agctgctgca g           - #                  - #                      - #21                                                                   - -  - - <210> SEQ ID NO 4                                                   <211> LENGTH: 33                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: primer         - - <400> SEQUENCE: 4                                                         - - ccaggccctt ctgggaggcc gctgtcacgg cgg       - #                  - #             33                                                                      - -  - - <210> SEQ ID NO 5                                                   <211> LENGTH: 33                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: primer         - - <400> SEQUENCE: 5                                                         - - gccgtgacag cggcctccca gaagggcctg gac       - #                  - #             33                                                                      - -  - - <210> SEQ ID NO 6                                                   <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: primer         - - <400> SEQUENCE: 6                                                         - - ctgggaactg ggaagctg             - #                  - #                      - #  18                                                                 __________________________________________________________________________

What is claimed are:
 1. An isolated polynucleotide encoding a bactericidal/permeability-increasing protein (BPI) deletion analog consisting of amino acid residues 10 through 193 of mature human BPI (SEQ ID NO:2), wherein a cysteine residue at position 132 is replaced by a different amino acid and wherein the amino acid at position number 185 is selected from the group consisting of lysine and glutamic acid.
 2. The polynucleotide of claim 1 wherein said cysteine residue at position 132 is replaced by alanine.
 3. The polynucleotide of claim 1 further comprising nucleotides encoding the twenty-seven amino acid leader sequence of BPI (amino acid residues -27 through -1 of SEQ ID NO: 2).
 4. The polynucleotide of claim 1 which is a DNA.
 5. An expression vector comprising the DNA according to claim
 4. 6. A host cell stably transformed or transfected with the DNA of claim 4 in a manner allowing expression in said host cell of said polypeptide deletion analog.
 7. A eukaryotic host cell according to claim
 6. 8. The host cell of claim 7 which is a CHO cell.
 9. A method for producing a BPI deletion analog polypeptide comprising growing a host cell according to claim 6 in a suitable culture medium and isolating said polypeptide from said host cell or said culture medium.
 10. The polynucleotide of claim 1 wherein said cysteine residue at position 132 is replaced by a nonpolar amino acid selected from the group consisting of alanine and serine. 